Analysis
qPCR ΔΔCt
Relative gene expression by the delta delta Ct method straight from your instrument export, with automatic column mapping, optional efficiency correction, replicate error propagation and a log-scale chart.
Raw data
Calculation settings
Amplification efficiency correction (Pfaffl)
At 100 per cent for both genes the result equals 2 to the power of minus delta delta Ct exactly. Take efficiencies from your own standard curve slope.
Results
The reported range propagates the standard deviation of technical replicates for the target and reference genes; it is not a substitute for a statistical test across biological replicates. Your file is never uploaded — everything is computed in the browser.
Formula
Fold = 2^(−ΔΔCt)
- ΔCt
- Ct of the target minus Ct of the reference gene, in the same sample
- ΔΔCt
- ΔCt of the sample minus ΔCt of the calibrator
- Fold
- relative expression; one means level with the calibrator
- Pfaffl
- when efficiency is not 100%: ratio = E_target^ΔCt_target ÷ E_ref^ΔCt_ref
Worked example
IL6 expression in a treated sample against a control, with GAPDH as reference
- Control: GAPDH = 20.0, IL6 = 25.0
- Treated: GAPDH = 20.0, IL6 = 23.0
- ΔCt control: 25.0 − 20.0 = 5.0
- ΔCt treated: 23.0 − 20.0 = 3.0
- ΔΔCt: 3.0 − 5.0 = −2.0
- Relative expression: 2 to the power of positive 2
Answer a fourfold increase — the calibrator always comes out at exactly one
Common mistakes
- Choosing a reference gene that itself responds to the treatment. GAPDH and beta-actin shift under hypoxia, differentiation and many treatments; validate reference stability in your own conditions.
- Using delta delta Ct when the two genes amplify with different efficiencies. The method assumes both double every cycle; if your standard curve says otherwise, enter the efficiencies in the advanced section of this tool.
- Reporting technical replicates as if they were biological ones. Three wells from one sample show pipetting precision, not real variation between organisms or cultures.
- Treating Undetermined as zero. That well means the target never crossed the threshold; substituting zero badly distorts the result. This tool drops such wells instead.
- Averaging fold changes rather than averaging ΔΔCt. Because the relationship is exponential, the order of those two operations changes the answer.
Frequently asked
How do I measure amplification efficiency?
Run a dilution series of cDNA and fit a standard curve. Efficiency is ten to the power of minus one over the slope, minus one. A slope near −3.32 means 100 per cent.
How many reference genes do I need?
Careful work uses at least two and takes their geometric mean. With a single reference, any drift in that one gene passes straight into your result.
What should I do about Ct above 35?
Starting copy numbers there are very low and replicate scatter grows. The Ct cut-off in this tool defaults to 38; lower it if your data are noisy.
What does the reported range mean?
It propagates the standard deviation of technical replicates for the target and reference genes. It is not a substitute for a statistical test across biological replicates and should not be reported as a confidence interval.